All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Chronic hepatitis is inflammation of the liver that lasts at least six months. Chronic hepatitis, although much less common than acute hepatitis, can persist for years, even decades. In most people, it is quite mild and does not cause significant liver damage. However, in some people, continued inflammation slowly damages the liver, eventually resulting in cirrhosis (severe scarring of the liver), liver failure, and sometimes liver cancer. Chronic hepatitis is usually caused by Hepatitis B virus (HBV) Hepatitis C virus (HCV) and drugs.
Chronic Hepatitis C virus (HCV) infection is characterized by the inability of the host to establish an effective immune response. At the same time chronic HCV infection is associated with persistent, abnormally high levels of immune activation. This activation contributes to liver damage and disease progression. Patients with chronic HCV infection have widely varying clinical courses, while some develop cirrhosis, others do not show progression of liver disease.
Several risk factors including concomitant ethanol consumption have been associated with accelerated liver damage and progression to cirrhosis. Patients with both chronic HCV and ethanol consumption have been found to have accelerated progression of liver disease. This has been associated with increased levels of LPS (Bedogni, G. Am. J. Gastroenterol. 103(9): 2248-53 (2008)). Interestingly, a recent study has shown that the HCV nonstructural protein NS5A activates Toll-like receptor (TLR) 4 which is also activated by LPS (Machida, K. et al. Proc. Natl. Acad. Sci. USA, 106(5):1548-53 (2009).
LPS may not only be connected with progression of liver disease, but also with the perpetuation of chronic infection. When compared with patients with self limiting infection, monocytes from patients with chronic HCV produce significantly more IL-10 and TNF alpha in response to the HCV core protein or LPS (Martin-Blondel, G. et al. J. Viral. Hepatitis (Mar. 11, 2009)). Thus, although translocation of gut microbial products, immune activation and progression of liver disease appear to be closely linked, proof of causality is lacking. Additionally, there appears to be a connection between LPS levels and viral clearance, though this needs to be elucidated. Thus, studies designed at clarifying these relationships are needed. If microbial translocation is driving immune activation and progression of liver disease, strategies that reduce or prevent microbial translocation may therefore have a significant impact on immune activation, and thus on the natural history of chronic HCV infection.
Spontaneous bacterial peritonitis (SBP) is a common and severe complication of chronic liver diseases, such as liver cirrhosis, portal hypertension and ascites. SBP occurs in up to 30% of patients, and is associated with an in-hospital mortality rate of up to 25%. Bacterial translocation into the stagnant and immune depleted peritoneal fluid is considered to be the main pathogenic mechanism of SBP. While paracentesis and broad spectrum antibiotic therapy constitute an effective treatment for acute infection, many patients suffer from recurrent episodes of SBP with pathogens which become increasingly resistant to antibiotic therapy (Song, K. H. et al. BMC Infect Dis. 9(1): 41 (2009)). Methods for SBP prophylaxis using chronic antibiotics are controversial and associated with immergence of antibiotic resistant species (Cohen, M. J. et al. Cochrane Database Syst Rev. 2: CD004791 (2009)).
Recently, an increasing association has been found between bacterial translocation and the incidence of complications of cirrhosis. The levels of either bacterial products (ribosomal 16s RNA) in the serum or endotoxemia (LPS or LBP) have been correlated with variceal bleeding, hepatorenal syndrome and the hyperdynamic circulatory state found in cirrhotic patients (El-Naggar, M. M. et al. J. Med. Microbiol. 57(Pt 12):1533-8 (2008)).
For decades, various attempts have been made to obtain increased secretion of immunogen-specific antibodies via the mammary gland of farm animals. Such attempts are aimed at production of large quantities of immunogen-specific antibodies via milk. The antibody levels in mature milk, however, still remain low (approximately an order of magnitude) when compared to those that can be achieved in colostrum.
Colostrum (also known as first milk) is a form of milk produced by the mammary glands in late pregnancy and the few days after birth. In humans it has high concentrations of nutrients and antibodies, but it is small in quantity. Colostrum is high in carbohydrates, protein, mineral salts, vitamins and immunoglobulin. It also contains various floating cells such as granular and stromal cells, neutrophils, monocyte/macrophages and lymphocytes and includes growth factors, hormones and cytokines.
Leukocytes are also present in colostrum in large numbers which enable protection against viruses and bacteria. Colostral leukocytes enhance passive immunity of neonatal calf, especially in regard to antibodies and immunoglobulin classes which are essential for intestinal immunity.
The large numbers of secretory antibodies found in the colostrum help protect the mucous membranes in the throat, lungs, and intestines of the newborn. Bovine colostrum (BC) contains three major classes of immunoglobulins: IgG, IgM and IgA.
As indicated above, colostrum is quite a unique product that arises from a distinct physiological and functional state of the mammary gland. In ruminants, the principal compositional difference between colostrum and mature milk is the very high content of bioactive components such as lactoferrin and immunoglobulins (Tarbell, K. V. et al. J. Exp. Med. 199:1467-77 (2004); Bluestone, J. A. and Tang, Q. J. Autoimmun 24:55-62 (2005); Putnam, A.l. et al J. Autoimmun. 24:55-62 (2005)), of which IgG class makes up 80-90%.
The immunization of an animal such as a cow with specific antigens enables the production and harvest of specific antibodies that may be used for modulation of an immune response and thereby in the treatment of immune-related disorders. Accordingly, this method serves as an easy and safe means for generating antigen-specific antibodies and immune adjuvants.
Several previous patents and patent applications by some of the present inventors, described the use of specific bacterial pathogens antibodies, obtained from bovine colostrum for the passive treatment of infectious diseases. For example, WO 04/078209 by some of the present inventors describes compounds and compositions for the treatment or prophylaxis of gastrointestinal disorders prepared by immunizing a host animal with a vaccine comprising one or more cell wall antigens of enteric bacteria, specifically, gram negative bacteria. The hyper immune material produced is in the form of tablets for oral administration. WO 03/097094 describes the use of a hyper immune colostrum in the production of antibodies (whole IgG), or F(ab′)2 antibodies fragments, conjugated with mammalian colostrum and colostrum extracts, for intranasal administration aimed at the prevention of symptoms arising from the presence of air-borne pathogenic bacteria.
Mucosal tolerance is considered as an attractive approach for the treatment of autoimmune and inflammatory diseases due to the lack of toxicity, ease of administration, and antigen-specific mechanism of action (Wershil, B. K. and Furuta, G. T. J. Allergy Clin. Immunol. 121:S380-3; quiz S415 (2008); Faria, A. M. and Weiner, H. L. Clin. Dev. Immunol. 13:143-57 (2006)). Hence, major attempts were made to generate stable colostrum-derived products suitable for oral and nasal administration. For Example, WO 95/08562 by some of the inventors, describes the method of obtaining high purity immunoglobulins from antibody rich colostrum and the possibility of compressing these colostral-antibodies into a tablet form without substantial loss of activity. Specific antibodies may be obtained by immunization of a mammal with specific antigens against enterotoxic bacteria such as E. coli, Salmonela and Shigella. WO 06/053383 by some of the inventors, describes a carboxylic acid and alkalizing moieties which confer upon a bioactive agent composition of a hyper immune colostrum, lactoferrin or lactoferracin, stability under a wide variety of gastric pH values. Finally, WO 03/080082 by some of the inventors describes a method of improving the viability of a labile bioactive substance, preferably immunoglobulins or fragments thereof or enzymes, in a gastric environment, comprising forming a mixture of the bioactive substance and mammalian colostrum and colostrums extracts. This conjugation protects the antibodies or antibodies fragments from the proteolysis occasioned by enzyme or low pH conditions and preserves their function in the stomach or rumen or other hostile environment.
The bowel mucosa is the largest lymphoid organ of the body. It deals with the dual role of nutrient absorption, while maintaining a physical and immunological barrier to the gut content. Despite constant antigenic stimulation, suppression of inflammation is the rule. Two key concepts pertain to the treatment of viral disease and its complications with colostrums: mucosal microbial translocation and enhanced immune regulation by oral feeding of disease antigens, termed “oral tolerance”.
Increased mucosal microbial translocation: this is an immerging concept in disease pathogenesis. The higher levels of microbial translocation, quantified by the presence of LPS and bacterial DNA are central to a state of chronic immune activation accounting for immune exhaustion and autoimmune damage.
Stimulation through the bowel mucosa tends to elicit a tolerogenic immune response. This feature may be used advantageously to induce tolerance towards auto-antigens and in this way to suppress autoimmunity. Indeed, “oral tolerance” has been shown to effectively diminish the immune response towards orally fed antigens in different disease models (Safadi, R. et al. Am. J. Gastroenterol. 98(11): 2505-15 (2003)).
It has been previously shown that bovine-derived colostrum preparations can be used in treating toxin-mediated intestinal conditions. In a study of 10 volunteers challenged orally with a concentrate of enterotoxigenic E. coli, administration of a bovine antibody concentrate obtained by immunizing cows with the corresponding E. coli strains prevented the development of diarrhea in all 10 participants who received the product; by contrast, 9/10 controls developed diarrhea (Tacket, C. O. et al. N. Engl. J. Med. 318(19): 1240-3 (1988)). In another study, the administration of milk-derived antibodies against the enterotoxigenic E. coli colonization factor protected 14/15 subjects from diarrhea, compared to 7/10 subjects given placebo (Freedman, D. J. et al. J. Infect. Dis. 177(3): 662-7 (1998)).
Another disease with a similar pathogenesis is pseudomembranous colitis. A study evaluated to effect of immune whey protein, obtained by immunizing cows with C. difficile inactivated toxins and whole-cell killed C. difficile shown as preventing relapse of C. difficile disease. Sixteen patients received the product after standard treatment for a confirmed episode of C. difficile colitis for two weeks. In all but one case, C. difficile toxin disappeared from the stool, and there were no recurrences after a median follow-up of 333 days (van Dissel, J. T. et al. J. Med. Microbiol. 54(2): 197-205 (2005)).
Collectively, these observations suggest that bovine-derived colostrum preparations deliver biologically active concentrations of specific antibodies to the intestinal lumen when taken orally, and might be capable of blocking various forms of bacterial toxins in the gut by that mechanism.
Since microbial translocation is driving immune activation and progression of liver disease, strategies that reduce or prevent microbial translocation may have a significant impact on immune activation, and thus on the natural history of chronic HCV infection. The present invention now demonstrates the use of bovine colostrum powder (BPC) preparations from immunized cows, containing high levels of antibodies, as immuno-modulators capable of reducing immune activation in response to microbial products such as LPS. Without being bound to any theory, the inventors hypothesize that the attachment of the BPC antibodies to the microbial antigens may prevent their translocation onto the blood stream, thereby restricting the immune response. These effects upon the immune system enable the use of such colostrum preparations for the treatment of infectious disease, which involve the immune system. More specifically, the present invention provides the use of a colostrum-derived preparation, comprising high concentrations of anti-LPS antibodies, in the treatment and amelioration of chronic liver diseases.
Microbial translocation is also associated with alteration of the liver inflammation in different liver disorders, including viral mediated, drug mediated, non-alcoholic steatohepatitis and any other hepatic disorder. Microbial translocation may also be associated with insulin resistance, diabetes type 2, obesity and overweight. As shown by the invention, prevention of such translocation may be achieved using the anti-LPS enriched colostrum of the invention, optionally along with regulation of regulatory T cells, or any other component of the immune system, using a combination of the anti-LPS enriched colostrum with colostrum preparations enriched with antibodies recognizing disease-specific antigens, for example, anti-insulin enriched colostrum. Thus, the invention further provides compositions, combined compositions and methods for the treatment of any acute or chronic liver disease, diabetes and any complication associated therewith, fatty liver, non-alcoholic steatohepatitis, and obesity.
It is therefore an object of the invention to provide the use of colostrum- or avian egg derived anti-LPS enriched immunoglobulin preparations in compositions and methods of treating, delaying or preventing the progression of chronic liver disease, cirrhosis and any complication or disorder associated therewith.
Another object of the invention is to provide combined compositions comprising a combination of anti-LPS antibodies enriched colostrum and antibodies recognizing at least one antigen specific for a pathologic disorder and uses thereof in the treatment of immune-related disorders.
These and other objects of the invention will become clearer as the description proceeds.